Conjugates of insulin-like growth factor binding protein-4 and poly (ethylene glycol)

ABSTRACT

A conjugate consisting of insulin-like growth factor binding protein 4 (IGFBP-4) and one or two polyethylene glycol) group(s), said polyethylene glycol) group(s) having an overall molecular weight of from about 30 to about 40 kDa is disclosed. This conjugate is useful for the treatment of cancer.

This invention relates to conjugates of insulin-like growth factorbinding protein-4 (IGFBP-4) with poly(ethylene glycol) (PEG),pharmaceutical compositions containing such conjugates, and methods forthe production and methods of use of such conjugates.

BACKGROUND OF THE INVENTION

The insulin-like growth factors (IGFs) are mitogens that play a pivotalrole in regulating cell proliferation, differentiation and apoptosis.Six IGF-binding proteins (IGFBPs) can influence the actions of IGFs (Yu,H., and Rohan, T., J. Natl. Cancer Inst. 92 (2000) 1472-1489).

Mature human IGFBP-4 is described in the literature as a monomericprotein of 24 kDa and consists of 237 amino acids. The molecular weightof the protein calculated from the amino add sequence is 26 kDa. Itsbiological role is reviewed in Yu, H., and Rohan, T., J. Natl. CancerInst. 92 (2000) 1472-1489. Conover, C. A., et al., in J. Biol. Chem. 270(1995) 4395-4400, describe protease-resistant mutants of IGFBP-4. Allfour IGFBP-4 mutants around the putative cleavage site at Met135-Lys136and the wildtype protein bind IGFs with equivalent affinities.Resistance of IGFBP-4 to proteolytic cleavage is also achieved bydeletion of amino acids 121-141 as described by Miyakoshi, N., et al. inEndocrinology 142 (2001) 2641-2648. Byun, D., et al., in J.Endocrinology 169 (2001) 135-143, determined several regions involved inIGF binding by IGFBP-4. Deletion of segments Leu72-Ser 91 or Leu72-His74results in loss of IGF binding. Also mutation of certain cysteineresidues in the N- and the C-terminal domain significantly reduces thebinding of IGFs.

IGFBP-4 was first isolated from medium conditioned by human osteosarcomeTE-89 cells (Mohan, S., et al., Proc. Natl. Acad. Sci. USA 86 (1989)8338-8342). IGFBP-4 is known to exist naturally as a non-glycosylatedform with an apparent molecular weight of 24 kDa or in the glycosylatedform weighing 28 kDa. Recombinant IGFBP-4 was produced by expression inseveral eukaryotic and prokaryotic systems. Human IGFBP-4 was producedby expression in E. coli as a fusion protein with glutathioneS-transferase (Honda, Y., et al., J. Clin. Endocrinol. Metab. 81 (1996)1389-1396) or as a fusion protein with a hexahistidine tag (Qin, X, etal., J. Biol. Chem. 273 (1998) 23509-23516) or by expression in yeast asubiquitin fusion protein (Kiefer, M. C., et al., J. Biol. Chem. 267(1992) 12692-12699). The sequence of human IGFBP-4 is described indetail in the SwissProt Database (http://www.expasy.ch) and identifiedby the Accession No. P 22692. The amino acid positions described in thefollowing refer to the sequence of the mature forms of IGFBP-4 (sequenceafter removal of the signaling peptide starts with amino acid inposition 1) or refer to the numbering used in the cited references.

IGFBP-4 inhibits the in vitro IGF-stimulated bone cell proliferation(Mohan, S., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 8338-8342), theIGP-mediated growth of chick cartilage (Schiltz, P. M., et al., J. BoneMineral Res. 8 (1993) 391-396) and the growth of HT-29 cells (Culouscou,J. M., and Shoyab, M., Cancer Res. 51 (1991) 2813-2819). Overexpressionof IGFBP-4 in the malignant M 12 prostate epithelial cell line reducesthe IGF-induced proliferation of the cells, inhibits colony formation insoft agar, increases apoptosis in response to 6-hydroxyurea and delaysthe formation of tumors by the transformed cells (Damon, S. E., et al.,Endocrinology 139 (1998) 3456-3464). Overexpression of IGFBP-4 in humancolorectal carcinoma cells reduces the proliferation of these cells andsuppresses colony formation (Diehl, D., et al., J. Cancer Res. ClinicalOncol. 127, Suppl.1 (2001) S54).

Covalent modification of proteins with poly(ethylene glycol) (PEG) hasproven to be a useful method to extend the circulating half-lives ofproteins in the body (Hershfield, M. S., et al., New England Journal ofMedicine 316 (1987) 589-596; and Meyers, F. J., et al., Clin. Pharmacol.Ther. 49 (1991) 307-313). Other advantages of PEGylation are an increaseof solubility and a decrease in protein immunogenicity (Katre, N. V., J.Immunol. 144 (1990) 209-213). A common method for the PEGylation ofproteins is the use of poly(ethylene glycol) activated withamino-reactive reagents like N-hydroxysuccinimide (NHS). With suchreagents poly(ethylene glycol) is attached to the proteins at freeprimary amino groups such as the N-terminal α-amino group and theε-amino groups of lysine residues. However, a major limitation of thisapproach is that proteins typically contain a considerable amount oflysine residues and therefore the poly(ethylene glycol) groups areattached to the protein in a non-specific manner at all of the freeε-amino groups, resulting in a heterologous product mixture of randomPEGylated proteins. Therefore, many NHS-PEGylated proteins areunsuitable for commercial use because of low specific activity.Inactivation results from covalent modification of one or more lysineresidues or the N-terminal amino residue required for biologicalactivity or from covalent attachment of the poly(ethylene glycol)residues near or at the active site of the protein. For example, it wasfound that modification of human growth hormone using NHS-PEGylationreagents reduces the biological activity of the protein by more than10-fold (Clark, R., et al., J. Biol. Chem. 271 (1996) 21969-21977).Human growth hormone contains 9 lysines in addition to the N-terminalamino acid. Certain of these lysines are located in regions of theprotein known to be critical for receptor binding (Cunningham, B. C., etal., Science 254 (1991) 821-825). In addition, the modification oferythropoietin by the use of amino-reactive poly(ethylene glycol)reagents results also in a nearly complete loss of biological activity(Wojchowski, D. M., et al., Biochim. Biophys. Acta 910 (1987) 224-232).Covalent modification of Interferon-α2 with amino-reactive PEGylationreagents results in 40-75% loss of bioactivity (U.S. Pat. No.5,382,657). A similar modification of G-CSF results in greater than 60%loss of activity (Tanaka, H., et al., Cancer Res. 51 (1991) 3710-3714)and of Interleukin-2 in greater than 90% loss of bioactivity (Goodson,R. J., and Katre, N. V., BioTechnology 8 (1990) 343-346).

Van den Berg, C. L., et al. (Europ. J. Cancer 33 (1997) 1108-1113; andWO 94/22466) covalently coupled cysteine-reactive poly(ethylene glycol)(20 kDa) to IGFBP-1, which leads to a prolonged serum half-life of 13.6h. As described in WO 94/22466 it is believed that amino acids in themiddle domain of IGFBP-1 can be substituted by cysteine for specificPEGylation without interference with the IGF binding and inhibition.Amino adds in positions 98 and 101 were exchanged against cysteinebecause Serine 101 is a natural major phosphoylation site, exposed onthe protein surface and not involved in binding to IGFs. The 20 kDamonoPEGylated IGFBP-1 shows a comparable but no improved in vitroactivity to wild-type IGFBP-1 regarding inhibition of tumor cell growth.According to van den Berg, C. L, et al., Europ. J. Cancer 33 (1997)1108-1113, their PEGylated IGFBP-1 in vivo may not be able to inhibitIGF action directly on the tumor cell.

It is an object of the present invention to provide an improved IGFBPderivative with inhibitory efficacy on tumor growth and prolonged halflife in vivo, which can preferably be administered as only a few bolusapplications per week and which is capable of suppressing tumor growth,angiogenesis and/or metastasis.

SUMMARY OF THE INVENTION

It has surprisingly been found that 30 kDa to 40 kDa PEGylated (30-40kDa PEGylated IGFBP-4), preferably monoPEGylated, IGFBP-4 according tothe invention has superior properties in regard to therapeuticapplicability in tumor treatment such as suppressing tumor growth,angiogenesis and/or metastasis in vivo, which cannot be found forIGFBP-4 alone or for lower weight PEGylated IGFBP-4. In addition, theconjugates according to the invention avoid undesired side effects invivo such as alteration of normal kidney cells found for lower weightPEGylated IGFBP-4.

The present invention provides a conjugate consisting of an insulin-likegrowth factor binding protein-4 (IGFBP-4) and one or two poly(ethyleneglycol) group(s), said poly(ethylene glycol) group(s) having an overallmolecular weight of from about 30 to 40 kDa. Preferably, thepoly(ethylene glycol) group(s) are conjugated to IGFBP-4 via primaryamino group(s) (amino-reactive PEGylation). It is also preferred thatthe conjugate is a monoPEG-IGFBP-4 conjugate. It is particularlypreferred that the conjugate is a mono-N-terminal PEG-IGFBP-4 conjugatecoupled via the N-terminal amino group of IGFBP-4.

Also preferred are conjugates that include a branched PEG.

The invention further comprises methods for the production of theconjugates according to the invention.

The invention further comprises pharmaceutical compositions containing aconjugate according to the invention.

The invention further comprises methods for the production ofpharmaceutical compositions containing a conjugate according to theinvention.

The invention further comprises the use of a conjugate according to theinvention for the preparation of a medicament for the treatment ofcancer, preferably pancreatic cancer.

The invention further comprises methods for the treatment of humancancer (e.g. breast, lung, prostate or colon cancer), preferablypancreatic cancer, characterized in that a pharmaceutically effectiveamount of 30-40 kDa PEGylated IGFBP-4 is administered to a patient inneed of such treatment, preferably in one to seven bolus applicationsper week.

DETAILED DESCRIPTION OF THE INVENTION

“Amino-reactive PEGylated IGFBP-4” or “amino-reactive PEGylation” asused herein means that IGFBP-4 is covalently bonded to one, two or threepoly(ethylene glycol) groups by amino-reactive coupling to the IGFBP-4molecule. The PEG groups can be attached at different sites of theIGFBP-4 molecule that are primary amino groups, preferably at the mostreactive sites, e.g., the ε-amino groups of the lysine side chains orthe N-terminal α-amino group. Due to the synthesis method used,PEGylated IGFBP-4 can consist of a mixture of mono- and/or diPEGylatedIGFBP-4, whereby the sites of PEGylation can be different in differentmolecules or can be substantially homogeneous in regard to the amount ofpoly(ethylene glycol) side chains per molecule and/or the site ofPEGylation in the molecule.

Amino-reactive PEGylation as used herein designates a method of randomlyattaching poly(ethylene glycol) chains to primary amino group(s) of thetarget protein IGFBP-4 by the use of reactive (activated) poly(ethyleneglycol), preferably by the use of N-hydroxysuccinimidyl esters of,preferably, methoxypoly(ethylene glycol). The coupling reactionpreferentially attaches poly(ethylene glycol) to reactive primary aminogroups like the ε-amino groups of lysine residues or the α-amino groupof the N-terminal amino acid of IGFBP-4. Such amino group conjugation ofPEG to proteins is well known in the art. For example, review of suchmethods is given by Veronese, F. M., Biomaterials 22 (2001) 405-417.According to Veronese, the conjugation of PEG to primary amino groups ofproteins can be performed by using activated PEGs which perform analkylation of said primary amino groups. For such a reaction, activatedalkylating PEGs, for example PEG aldehyde, PEG-tresyl chloride or PEGepoxide can be used. Further useful reagents are acylating PEGs such ashydroxysuccinimidyl esters of carboxylated PEGs or PEGs in which theterminal hydroxy group is activated by chloroformates orcarbonylimidazole. Further useful PEG reagents are PEGs with amino acidarms. Such reagents can contain the so-called branched PEGs, whereby atleast two identical or different PEG molecules are linked together by apeptidic spacer (preferably lysine) and, for example, bound to IGFBP-4as activated carboxylate of the lysine spacer. Mono-N-terminal couplingis also described by Kinstler, O., et al., Adv. Drug Deliv. Rev. 54(2002) 477-485.

In the discussion and examples below, some preferred reagents for theproduction of amino-reactive PEGylated IGFBP-4 are described. It isunderstood that modifications, for example, based on the methodsdescribed by Veronese, F. M., Biomaterials 22 (2001) 405-417, can bemade in the procedures as long as the process results in conjugatesaccording to the invention.

The occurrence of several potentially reactive primary amino groups inthe target protein (for IGFBP-4 there are 12 lysines+1 terminal aminoacid) leads to a series of PEGylated IGFBP-4 isomers that differ in thepoint of attachment of the poly(ethylene glycol) chain and willhereinafter be referred to as “positional isomers”. The attachment sitein a single IGFBP-4 molecule is not clearly predicted and for thatreason referred to as “random”. Nine of these twelve lysines (Lys 67,Lys 124, Lys 134, Lys 136, Lys 192, Lys 204, Lys 210, Lys 215 and Lys223) are located in regions that are reported to be required for complexformation with IGF (Qin, X., et al., J. Biol. Chem. 273 (1998)23509-23516). Therefore, a strong reduction of the affinity to IGF wouldbe expected for randomly PEGylated IGFBP-4. Surprisingly, this was notthe case for the conjugates according to the invention.

It is also preferred to attach the PEG groups to IGFBP-4 viathiol-reactive PEGylation. Thiol-reactive PEGylation as used hereindesignates a method of attaching poly(ethylene glycol) to a targetprotein (IGFBP-4 mutant) by the use of activated, thiol-reactivepoly(ethylene glycol), preferably by the use of N-maleimide esters of,preferably, methoxypoly(ethylene glycol). The coupling reactionpreferentially attaches poly(ethylene glycol) to Cys110 and/or Cys117.Such sulfhydryl conjugation of PEG to proteins is widely known in thestate of the art. A review of such methods is also given by, forexample, Veronese, F. M., Biomaterials 22 (2001) 405-417. According toVeronese, the conjugation of PEG to thiol groups of proteins can beperformed by using thiol-activated PEGs. For such a reaction, activatedthiol-reactive PEGs, for example PEG-orthopyridyl-disulfide,PEG-maleimide, PEG-vinylsulfone, and PEG-iodoacetamide, can be used.

The invention provides PEGylated forms of IGFBP-4 with improvedproperties. Such PEGylated IGFBP-4 conjugates contain one or two PEGgroups linear or branched and randomly attached thereto, whereby theoverall molecular weight of all PEG groups in the conjugate is about 30to 40 kDa It is obvious to a person skilled in the art that smalldeviations from this range of molecular weight are possible as long asthe PEGylated IGFBP-4 does not show such a negative influence on normalkidney cells as described in Example 15 for PEG₂₀-IGFBP-4. AlsoPEGylation of IGFBP-4 with PEG having molecular weights of more than 40kDa results in antitumorigenic activity. However, it is expected thatsuch activity decreases as the molecular weight increases due to reducedtumor penetration. Therefore, the range of 30 to 40 kDa for themolecular weight of PEG has to be understood as the optimized range fora conjugate of PEG and IGFBP-4 useful for an efficient treatment of apatient suffering from a cancerous disease.

As used herein, “molecular weight” means the mean molecular weight ofthe PEG. The term “about” before a designated molecular weight indicatesthat in said PEG preparations, some molecules will weigh more and someless than the stated molecular weight.

The following PEGylated forms of IGFBP-4 are examples of and arecontemplated embodiments of the conjugates of the invention:

-   -   monoPEGylated IGFBP-4, wherein the PEG group has a molecular        weight of 30 or 40 kDa;    -   diPEGylated IGFBP-4, wherein the PEG groups have a molecular        weight of about 20 kDa each;    -   and mixtures thereof.

“PEG or PEG group” according to the invention means a residue containingpoly(ethylene glycol) as an essential part. Such a PEG can containfurther chemical groups which are necessary for binding reactions; whichresults from the chemical synthesis of the molecule; or which is aspacer for optimal distance of the parts of the molecule from oneanother. In addition, such a PEG can consist of one or more PEGside-chains which are linked together. PEG groups with more than one PEGchain are called multiarmed or branched PEGs. Branched PEGs can beprepared, for example, by the addition of polyethylene oxide to variouspolyols, including glycerol, pentaerythriol, and sorbitol. For example,a four-armed branched PEG can be prepared from pentaerythriol andethylene oxide. Branched PEGs usually have 2 to 8 arms and are describedin, for example, EP-A 0 473 084 and U.S. Pat. No. 5,932,462. Especiallypreferred are PEGs with two PEG side-chains (PEG2) linked via theprimary amino groups of a lysine (Monfardini, C., et al., Bioconjug.Chem. 6 (1995) 62-69).

“Substantially homogeneous” as used herein means that the only PEGylatedIGFBP-4 molecules produced, contained or used are those having one ortwo PEG group(s) attached. The preparation may contain small amounts ofunreacted (i.e., lacking PEG group) protein. As ascertained by peptidemapping and N-terminal sequencing, one example below provides for thepreparation which is at least 90% PEG-IGFBP-4 conjugate (preferablymonoPEGylated) and at most 5% unreacted protein. Isolation andpurification of such homogeneous preparations of PEGylated IGFBP-4 canbe performed by usual purification methods, preferably size exclusionchromatography.

“MonoPEGylated” as used herein means that IGFBP-4 is PEGylated at onlyone amino group per IGFBP-4 molecule, whereby only one PEG group isattached covalently at this site and the sites of attachment can varywithin the monoPEGylated species. The monoPEGylated IGFBP-4 is at least90% of the preparation, and most preferably, the monoPEGylated IGFBP-4is 92%, or more, of the preparation, the remainder being unreacted(non-PEGylated) IGFBP-4. The monoPEGylated IGFBP-4 preparationsaccording to the invention are therefore homogeneous enough to displaythe advantages of a homogeneous preparation, e.g., in a pharmaceuticalapplication. The same applies to the diPEGylated species.

“Activated PEGs or activated PEG reagents” are well-known in the stateof the art. Preferably there are used electrophilically activated PEGssuch as alkoxybutyric add succinimidyl esters of poly(ethylene glycol)(“lower alkoxy-PEG-SBA”) or alkoxypropionic add succinimidyl esters ofpoly(ethylene glycol) (“lower alkoxy-PEG-SPA”) or N-hydroxysuccinimideactivated PEGs. Any conventional method of reacting an activated esterwith an amine to form an amide can be utilized. In the reaction of theactivated PEG with IGFBP-4, the exemplified succinimidyl ester is aleaving group causing the amide formation. The use of succinimidylesters to produce conjugates with proteins is disclosed in U.S. Pat. No.5,672,662.

When the PEGylation reagent is combined with IGFBP-4, it is found thatat a pH of about 7.0, a protein:PEG ratio of about 1:3, and a reactiontemperature of from 20-25° C., a mixture of mono-, di-, and traceamounts of the tri-PEGylated species are produced. When the protein:PEGratio is about 1:1, primarily the monoPEGylated species is produced. Bymanipulating the reaction conditions (e g., ratio of reagents, pH,temperature, protein concentration, time of reaction etc.), the relativeamounts of the different PEGylated species can be varied.

MonoPEGylated IGFBP-4 can also be produced according to the methodsdescribed in WO 94/01451. WO 94/01451 describes a method for preparing arecombinant polypeptide with a modified terminal amino acid alpha-carbonreactive group. The steps of the method involve forming the recombinantpolypeptide and protecting it with one or more biologically addedprotecting groups at the N-terminal alpha-amine and C-terminalalpha-carboxyl. The polypeptide can then be reacted with chemicalprotecting agents to selectively protect reactive side chain groups andthereby prevent side chain groups from being modified. The polypeptideis then cleaved with a cleavage reagent specific for the biologicalprotecting group to form an unprotected terminal amino add alpha-carbonreactive group. The unprotected terminal amino acid alpha-carbonreactive group is modified with the activated PEG reagents. The sidechain protected terminally modified single copy polypeptide is thendeprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification.

IGFBP-4 conjugates according to the invention may be prepared bycovalently reacting a primary amino group of an IGFBP-4 protein with abifunctional reagent to form an intermediate with an amide linkage andcovalently reacting the intermediate containing amide linkage with anactivated poly(ethylene glycol) derivative to form an IGFBP-4 proteinconjugate. In the foregoing process, the bifunctional reagent ispreferably N-succinimidyl-S-acetylthiopropionate orN-succinimidyl-S-acetylthioacetate, and the activated poly(ethyleneglycol) derivative is preferably selected from the group consisting ofiodo-acetyl-methoxy-PEG, methoxy-PEG-vinylsulfone, andmethoxy-PEG-maleimide.

The IGFBP-4 conjugates may be prepared by amino-reactive covalentlinking of thiol groups to IGFBP-4 (“activation”) and coupling theresulting activated IGFBP-4 with a poly(ethylene glycol) (PEG)derivative. The first step comprises covalent linking of thiol groupsvia NH₂-groups of IGFBP-4. This activation of IGFBP-4 is performed withbifunctional reagents which carry a protected thiol group and anadditional reactive group, such as active esters (e.g., asuccinimidylester), anhydrides, esters of sulphonic acids, halogenidesof carboxylic acids and sulphonic acids, respectively. The thiol groupis protected by groups known in the art, e.g., acetyl groups. Thesebifunctional reagents are able to react with the ε-amino groups of thelysine amino acids by forming an amide linkage. The preparation of thebifunctional reagents is known in the art. Precursors of bifunctionalNHS esters are described in DE 39 24 705, while the derivatization tothe acetylthio compound is described by March, J., Advanced OrganicChemistry (1977) 375-376. The bifunctional reagent SATA is commerciallyavailable (Molecular Probes, Eugene, Oreg., USA and Pierce, Rockford,Ill.) and described in Duncan, R. J., Anal. Biochem. 132 (1983) 68-73.

The number of thiol groups to be added to an IGFBP-4 molecule can beselected by adjusting the reaction parameters, i.e., the protein(IGFBP-4) concentration and the protein/bifunctional reagent ratio.Preferably, the IGFBP-4 is activated by covalently linking from 1 to 5thiol groups per IGFBP-4 molecule, more preferably from 1.5 to 3 thiolgroups per IGFBP-4 molecule. These ranges refer to the statisticaldistribution of the thiol group over the IGFBP-4 protein population.

The reaction is carried out, for example, in an aqueous buffer solution,pH 6.5-8.0, e.g., in 10 mM potassium phosphate, 300 mM NaCl, pH 7.3. Thebifunctional reagent may be added in DMSO. After completion of thereaction, preferably after 30 minutes, the reaction is stopped byaddition of lysine. Excess bifunctional reagent may be separated bymethods known in the art, e.g., by dialysis or column filtration. Theaverage number of thiol groups added to IGFBP-4 can be determined byphotometric methods described in, for example, Grasetti, D. R, andMurray, J. F. in J. Appl. Biochem. Biotechnol. 119 (1967) 41-49.

The above reaction is followed by covalent coupling of an activatedpoly(ethylene glycol) (PEG) derivative. Suitable PEG derivatives areactivated PEG molecules with an average molecular weight of from about15 to about 40 kDa, depending on whether mono- or diPEGylated product isdesired.

Activated PEG derivatives are known in the art and are described in, forexample, Morpurgo, M., et al. J. Bioconjug. Chem. 7 (1996) 363-368 forPEG-vinylsulfone. Linear chain and branched chain PEG species aresuitable for the preparation of the compounds of formula I. Examples ofreactive PEG reagents are iodo-acetyl-methoxy-PEG andmethoxy-PEG-vinylsulfone. The use of these iodo-activated substances isknown in the art and is described e.g. by Hermanson, G. T., inBioconjugate Techniques, Academic Press, San Diego (1996) p. 147-148.

A preferred method for cysteine specific PEGylation as used hereindesignates a method of attaching poly(ethylene glycol) chains to atarget polypeptide (IGFBP-4) by the use of a 20 kDamethoxy-poly(ethylene glycol)-maleimide or branched 40 kDaPEG2-maleimide (=PEG-maleimide) (Shearwater Polymers, Inc.; Huntsville,Ala.) to a reduced sufhydryl group of the polypeptide chain of theprotein. Native IGFBP-4 does not possess free cysteins because allcysteins are involved in the formation of disulfide bonds. Reduction ofnative IGFBP-4 in the presence of mild or low concentrations of reducingagents such as β-mercaptoethanol, dithiotreitol or TCEP results inselective opening of a disulfide bond and the exposure of reducedsulfhydryl groups which can be specifically modified with PEG-maleimide.

It is assumed that the disulfide bonds in the middle domain of IGFBP-4are highly sensitive to reduction and therefore enablescysteine-specific PEGylation at cysteine110 or cysteine117. Thespecificity of the coupling reaction for cysteine 110 and 117 of IGFBP-4was confirmed by peptide mapping of the isolated monoPEGylated IGFBP-4and identification of the peptides by LC-MS mass spectrometry andsequencing of the peptide peaks. PEGylated forms of IGFBP-4 demonstrateda reduced peak area of the peptide containing the two cysteines.

Most preferably, the PEG species are activated by maleimide using(alkoxy-PEG-maleimide), such as methoxy-PEG-maleimide (MW 15,000 to40,000; Shearwater Polymers, Inc.). The coupling reaction withalkoxy-PEG-maleimide takes place after in situ cleavage of the thiolprotecting group in an aqueous buffer solution, e g. 10 mM potassiumphosphate, 300 mM NaCl, 2 mM EDTA, pH 6.2. The cleavage of theprotecting group may be performed, for example, with hydroxylamine inDMSO at 25° C., pH 6.2 for about 90 minutes. For the PEG modificationthe molar ratio of activated IGFBP-4/alkoxy-PEG-maleimide should be fromabout 1:1 to about 1:6. The reaction may be stopped by addition ofcysteine and reaction of the remaining thiol (—SH) groups withN-methylmaleimide or other appropriate compounds capable of formingdisulfide bonds. Because of the reaction of any remaining active thiolgroups with a protecting group such as N-methylmaleimide or othersuitable protecting group, the IGFBP-4 proteins in the conjugates ofthis invention may contain such protecting groups. Generally theprocedure described herein will produce a mixture of molecules havingvarying numbers of thiols protected by different numbers of theprotecting group, depending on the number of activated thiol groups onthe protein that were not conjugated to PEG-maleimide.

Whereas N-methylmaleimide forms the same type of covalent bond when usedto block the remaining thiol-groups on the PEGylated protein, disulfidecompounds will lead in an intermolecular sulfide/disulfide exchangereaction to a disulfide bridged coupling of the blocking reagent.Preferred blocking reagents for that type of blocking reaction areoxidized glutathione (GSSG), cysteine and cystamine. Whereas withcysteine no additional net charge is introduced into the PEGylatedprotein, the use of the blocking reagents GSSG or cystamine results inan additional negative or positive charge.

Thiol-reactive PEGylation of IGFBP-4 mutants can be performed accordingto the methods of the state of the art (see, e.g., WO 94/22466,andVeronese, F. M., Biomaterials 22 (2001) 405-417). Further activated PEGderivatives are known in the art and are described in, for example,Morpurgo, M., et al. J. Bioconjug. Chem. 7 (1996) 363-368 forPEG-vinylsulfone. Linear chain and branched chain PEG species aresuitable for the preparation of the compounds of Formula 1. Examples ofreactive PEG reagents are iodo-acetyl-methoxy-PEG andmethoxy-PEG-vinylsulfone. The use of these iodo-activated substances isknown in the art and described e.g. by Hermanson, G. T., in BioconjugateTechniques, Academic Press, San Diego (1996) p. 147-148.

The further purification of the compounds according to the inventionincluding the separation of mono- and/or diPEGylated IGFBP-4 speciesfrom higher PEGylated forms maybe done by methods known in the art,e.g., column chromatography.

The percentage of mono-PEG conjugates as well as the ratio of mono- anddi-PEG species can be controlled by pooling broader fractions around theelution peak to decrease the percentage of mono-PEG or narrowerfractions to increase the percentage of mono-PEG in the composition.About ninety percent mono-PEG conjugates is a good balance of yield andactivity. Sometimes compositions in which, for example, at leastninety-two percent or at least ninety-six percent of the conjugates aremono-PEG species (n equals 1) may be desired. In an embodiment of thisinvention the percentage of conjugates where n is 1 is from ninetypercent to ninety-six percent.

Pharmaceutical Formulations:

PEGylated IGFBP-4 can be administered as a mixture, or as the ionexchange chromatography or size exclusion chromatography separateddifferent PEGylated species. The compounds of the present invention canbe formulated according to methods for the preparation of pharmaceuticalcompositions which methods are known to the person skilled in the art.For the production of such compositions, PEGylated IGFBP-4 according tothe invention is combined in a mixture with a pharmaceuticallyacceptable carrier, preferably by dialysis against an aqueous solutioncontaining the desired ingredients of the pharmaceutical compositions.Such acceptable carriers are described, for example, in Remington'sPharmaceutical Sciences, 18^(th) edition, 1990, Mack Publishing Company,edited by Oslo et al. (e.g. pp. 1435-1712). Typical compositions containan effective amount of the substance according to the invention, forexample from about 0.1 to 100 mg/ml, together with a suitable amount ofa carrier. The compositions may be administered parenterally.

The pharmaceutical formulations according to the invention can beprepared according to known methods in the art Usually, solutions ofPEGylated IGFBP-4 are dialyzed against the buffer intended to be used inthe pharmaceutical composition and the desired final proteinconcentration is adjusted by concentration or dilution.

Such pharmaceutical compositions may be used for administration forinjection or infusion and contain an effective amount of themonoPEGylated IGFBP-4 together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions include diluents of various buffer contents(e.g. arginine, acetate, phosphate), pH and ionic strength, additivessuch as detergents and solubilizing agents (e.g. Tween™ 80/polysorbate,pluronic™ F68), antioxidants (e.g. ascorbic acid, sodium metabisulfite),preservatives (Timersol™, benzyl alcohol) and bulking substances (e.g.saccharose, mannitol), incorporation of the material into particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic add, etc. or into liposomes. Such compositions mayinfluence the physical state stability rate of release and clearance ofthe monoPEGylated IGFBP-4 according to the invention.

Dosages and Drug Concentrations:

Typically, in a standard cancer treatment regimen, patients are treatedwith dosages in the range between 0.01 to 3 mg of PEGylated IGFBP-4 perkg per day over a certain period of time, lasting from one day to about30 days or even longer. Drug is applied as a singe daily subcutaneous ori.v. or i.p. (intraperitoneal) bolus injection or infusion of apharmaceutical formulation containing 0.1 to 100 mg PEGylated IGFBP-4per ml. This treatment can be combined with any standard (e.g.chemotherapeutic) treatment, by applying PEGylated IGFBP-4 before,during or after the standard treatment. This results in an improvedoutcome compared to standard treatment alone.

The following examples, references and figures are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: PEGylation of IGFBP-4 with 40 kDa PEG and separation of thePEGylated products by SEC. A) Coomassie stained SDS-PAGE of startingmaterial (lane 1) and outcome of the 40 kDa PEGylation reaction.Std=Mark12 Molecular weight standard (Invitrogen); 1=IGFBP-4 wildtype(starting material); 2=IGFBP-4 after PEGylation with 40 kDa mPEG2. B)Separation of the PEGylated products by size exclusion chromatography(“SEC”). SEC of random 40 kDa PEGylated IGFBP-4 was performed onSuperose 6 (Pharmacia) in 20 mM Phosphate pH7.5, 500 mM NaCl, flow rate0.5 ml/min. C) Analysis of PEGylated products by SDS PAGE. Std=Mark12Molecular weight standard (Invitrogen); 3=polyPEG40-IGFBP-4;4=unPEGylated IGFBP-4; 5=monoPEG40-IGFBP-4.

FIG. 2: Serum kinetics of mono40 kDa-PEG-IGFBP-4 compared with mono20kDa-PEG-IGFBP-4 and unPEGylated IGFBP-4. SCID mice were injectedsubcutaneously with a single dose of 1 mg/200 μl mono40 kDa-PEG-IGFBP-4or mono20 kDa-PEG-IGFBP-4 or unPEGylated IGFBP-4 in PBS. Serum sampleswere collected in a time range from 0.5 to 120 h after injection, asindicated, and analyzed for mono40 kDa-PEG-IGFBP-4 or mono20kDa-PEG-IGFBP-4 or unPEGylated IGFBP-4 by Western Blotting with ananti-IGFBP-4 antibody (UBI) after affinity purification or by ELISA.

FIG. 3: Inhibition of IGF-I mediated phosphorylation of IGF-I receptorby IGFBP-4 derivatives.

A) Western blot analysis of IGF-IR phosphorylation by IGF-I in theabsence and presence of IGFBP-4 derivatives. NIH3T3 cells overexpressingIGF-I-receptor were stimulated with 2 nM IGF-I in the presence orabsence of a threefold excess of IGFBP-4. After 10 minutes, cells werelysed and lysates subjected to a Western Blotting procedure withanti-phosphotyrosine antibodies to detect tyrosine phosphorylated IGF-Ireceptor. Mono20 kDa-PEG-IGFBP-4 and mono40 kDa-PEG-IGFBP-4 inhibitedthe receptor stimulation completely at concentrations of 6 nM. In termsof inhibiting IGF-I induced IGF-I-receptor phosphorylation bothPEGylated forms of IGFBP-4 proved to be as efficient as wildtypeIGFBP-4.

B) Titration of IGF-I mediated phosphorylation of IGF-I receptor byIGFBP-4 derivatives. NIH3T3 cells overexpressing IGF-I-receptor werestimulated with 3.3 nM IGF-I in the presence or absence of varyingconcentrations of IGFBP-4 derivatives. After 10 minutes, cells werelysed and lysates subjected to an ELISA based measurement ofphosphorylated IGF-I-Receptor. MonoPEGylated IGFBP-4 (20 or 40 kDa) andwildtype IGFBP-4 inhibited the receptor stimulation with an IC50 of 3nM.

FIG. 4: IGF-binding of monoPEG20-IGFBP-4 determined by Size exclusionchromatography. The binding abilities of IGFBP-4 or PEGylated isoformsthereof were determined by a size exclusion chromatography based assay.70 nmol (6 μg) of IGF-I are injected on the column (HRP 75, Pharmacia;running conditions: 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 1ml/min) either alone or together with 96 nmol mono20 kDa-PEG-IGFBP-4(equivalent to 25 μg wildtype IGFBP-4) after a preincubation step (30min at room temperature). Free IGF-I is quantified by integrating theIGF-I peak of the chromatogram (Chromeleon, Dionex). The peak area isnegatively correlated with the binding capacity of IGFBP-4. In thedemonstrated experiment, more than 90% of IGF-I is bound by mono20kDa-PEG-IGFBP-4. Similar results were obtained with mono40kDa-PEG-IGFBP-4.

FIG. 5: Inhibition of the IGF-I binding to immobilized wildtype IGFBP-4by wildtype, mono- and poly-20 kDa random PEGylated IGFBP-4.Determination of IC50 values of wildtype, monoPEG-IGFBP-4 andpolyPEG-IGFBP-4. For measuring IC50 values, wildtype IGFBP-4 wasimmobilized on a sensor chip surface. Binding of IGF-1 (10 nM) to theimmobilized IGFBP-4 was challenged by the addition of increasingconcentrations of wildtype IGFBP-4, monoPEG20-IGFBP-4 andpolyPEG20IGFBP-4 prior to chip contact Competition of IGF-I binding tothe immobilized IGFBP-4 with mono- or polyPEGylated IGFBP-4 was found tobe as efficient as with wildtype IGFBP. Similar results were obtainedwith 40 kDa-PEG-IGFBP-4.

EXAMPLE 1

Fermentation, Renaturation and Purification of IGFBP-4

Production of recombinant wildtype IGFBP-4 in E. coli or yeast wasdescribed for example by Miyakoshi, N., et al., Endocrinology 142(2001)2641-2648, and by Kiefer, M. C., et al., J. Biol. Chem. 267 (1992)12692-12699. Human recombinant IGFBP-4 is further commercially available(e.g. from GroPep Ltd.; Adelaide, Australia).

Fermentation Conditions:

Seed culture was performed in a 500 ml Erlenmeyer-flask with a culturevolume of 100 ml at 37° C. on a shaking incubator for 7 h. The mainculture was carried out in a 10 l fed-batch fermentation with an initialvolume of 81. The pH of the culture medium (Springer-Yeast 50 g/l,K₂HPO₄*₃H₂O₃ g/l, MgSO₄*₇H₂O 0.74 g/l, glucose 4.0 g/l, ampicillin 100mg/l, kanamycinsulfate 50 mg/l) was maintained at pH 6.8+0.3 by additionof an ammonia solution (12% w/v) as base and a glucosemonohydratesolution (75% w/v) as acid and carbon source. The dissolved oxygen levelwas sustained at or above 20% by supplying air at a rate of 1.0 vvm andaltering the agitation speed (500 rpm-1000 rpm).

After the culture reached an optical density (OD) of 10 (measured at 580nm with UV-visible spectrometer) the feeding with a Springer-yeastsolution (500 g/l) was started. The induction of the protein expressionwas carried out at an OD of 15 with 1 mMIsopropyl-β-D-thiogalactopyranoside (IPTG). IGFBP-4 is predominantlyexpressed insoluble as inclusion bodies (approx. 80%).

Cultivation was continued up to 12 h to an OD of 35 and then the cellswere harvested by centrifugation (13,000 rpm for 30 min).

Isolation and Purification of Inclusion Bodies:

The cell pellet was suspended in 0.1 M Tris-HCl buffer (pH 7.0)containing 1 mM MgSO₄. After addition of 0.3 g lysozyme per 100 g drycell weight and 30 U benzonase per 1 g dry cell weight, the cellsuspension was subjected to French press (14,500 psi, one cycle) fordisruption. After disruption the suspension was diluted 1:2 withBrij-buffer (200 ml/l Brij 30%, 1.5M NaCl, 0.1M EDTA, pH 7.0) andfurther stirred at room temperature for 30 minutes. To isolate theinclusion bodies the suspension was centrifuged at 13,000 rpm for 30min. The obtained pellet was resuspended with 0.1M Tris-HCl buffer(pH6.5) containing 5 mM EDTA and centrifuged again at 13,000 rpm for 30min. The pellet containing the purified inclusion bodies was stored at−20° C. until further purification of IGFBP-4.

Solubilization, Naturation and Purification:

20 g of inclusion bodies were solubilized in a buffer containing 8Mguanidinium chloride, 100 mM Tris, 5 mM EDTA and 100 mM DTE (pH 8.5).After solubilization, pH 2.5 was adjusted with HCl and the solubilisatewas dialyzed against 6M guanidinium chloride, 5 mM EDTA (pH 2.5).Protein content was analyzed by UV absorption. Naturation was performedat room temperature. The unfolded protein was diluted in two pulses(with a 5 h interval) of 0.25 mg protein per ml in a volume of 4500 ml(0.6M arginine, 1 mM EDTA, 3 mM GSH, 1 mM GSSG (pH8.5)) to a finalprotein concentration of 0.5 mg/ml. Naturation was completed over night.

The naturation sample was stocked up to 25% (NH₄)₂SO₄ and centrged. Thesupernatant was dialyzed against 50 mM sodium citrate, 100 mM NaCl (pH4.5) and brought to 0.8M (NH4)2SO4 and 0.2M arginine (by the addition ofsolid (NH₄)₂SO₄ and dilution of a 1M arginine/HCl stock solution). AfterpH adjustment to pH 8.5 with NaOH, the sample was applied to a phenylsepharose column (phenyl sepharose fast flow (Pharmacia); equilibratedwith 20 mM sodium phosphate, 100 mM NaCl, 1M (NH₄)₂SO₄ (pH 7.5)). Thecolumn was washed with equilibration buffer without (NH₄)₂SO₄. Elutionof IGFBP-4 was achieved in a gradient from 20 mM sodium phosphate to 20mM sodium phosphate supplemented with 50% ethylene glycol and apost-elution wash with 20 mM sodium phosphate, 100 mM NaCl, 50% ethyleneglycol, pH 7.5.

The eluate was pooled according to SDS-PAGE, diluted 1:2 with 50 mMcitrate pH 4.2 and applied on a S-sepharose column (Pharmacia). Thecolumn was washed with 20 mM sodium phosphate pH 7.5 and elution wasperformed with a gradient to 20 mM sodium phosphate, 600 mM NaCl IGFBP-4was finally pooled on the basis of SDS-PAGE.

EXAMPLE 2

40 kDa PEGylation of IGFBP-4 (Random Amino-Reactive PEGylation)

40 kDa PEGylation is achieved by reacting IGFBP-4 with mPEG2-NHS ester,a lysine derivative carrying two 20 kDa PEG chains and a single reactiveN-Hydroxysuccinimidyl ester (Shearwater Polymers, Inc.; Huntsville,Ala., USA; thereafter named 40 kDa mPEG2).

IGFBP-4 is PEGylated by the addition of an aqueous solution of 40 kDamPEG2 to a concentrated IGFBP-4 solution in PBS. 40 kDa mPEG2 was addedin a molar ratio of 2 molecules PEG per molecule IGFBP-4. The reactionwas allowed to proceed at room temperature for 30 minutes and wasfinally quenched by adding 1M arginine solution (buffered to pH 8.0 withHCl) to a final concentration of 100 mM.

The outcome of the PEGylation reaction was optimized for maximalproduction of monoPEGylated IGFBP-4 with simultaneous minimalconsumption of mPEG2-NHS reagent by carefully titrating protein and PEGconcentrations. For IGFBP-4, yields of the monoPEGylated isoforms isbest at elevated protein concentrations (c=5 mg/ml or higher) and a 2fold molar excess of PEGylation reagent.

EXAMPLE 3

N-terminal PEGylation of IGFBP-4

N-terminal specific PEGylation as used herein designates a method ofattaching poly(ethylene glycol) chains to a target polypeptide (IGFBP-4)by the use of a poly(ethylene glycol)aldehyde at acidic pH underreducing conditions. The coupling reaction preferentially attachesPEG-aldehyde to the N-terminal aminogroup of a polypeptide chain withlittle or no side reactions involving ε-amino groups of lysine.

Human IGFBP-4 was dialyzed against 20 mM acetate buffer, pH4.5 andPEGylated by the addition of an aqueous solution of 40 kDa or 20 kDaPEG-aldehyde (Shearwater Polymers, Inc.; Huntsville, Ala.). PEG-aldehydewas added in a molar ration of 2 molecules PEG per molecule IGFBP-4.PEG-aldehyde forms a Schiff base with the N-terminal amino group whichis subsequently (i.e. after one hour of incubation) reduced by theaddition of sodium cyano borohydrid to a final concentration of 20 mM.The reaction is allowed to proceed over night at room temperature.

The outcome of the PEGylation reaction was optimized for maximalproduction of N-terminally monoPEGylated IGFBP-4 with simultaneousminimal consumption of PEG-aldehyde reagent by carefully titratingprotein and PEG concentrations. For IGFBP-4, yields of the N-terminallymonoPEGylated isoforms are best at elevated peptide concentrations (c=1mg/ml or higher) and a 1.5 molar excess of PEGylation reagentPurification of monoPEGylated IGFBP-4 was performed as described for therandom PEGylated protein. The specificity of the coupling reaction forthe N-terminus of the protein was confirmed by N-terminal sequencing andpeptide mapping of the isolated monoPEGylated IGFBP-4 by use of theendoproteinase Lys C (sequence grade; Roche Diagnostics GmbH; Germany)and identification of the peptides by LC-MS mass spectrometry.

EXAMPLE 4

Cysteine Specific PEGylation of Wild-Type IGFBP-4

Cysteine specific PEGylation as used herein designates a method ofattaching poly(ethylene glycol) chains to a target polypeptide (IGFBP-4)by the use of a 20 kDa methoxy-poly(ethylene glycol)-maleimide orbranched 40 kDa PEG2-maleimide (=PEG-maleimide) (Shearwater Polymers,Inc.; Huntsville, Ala.) to a reduced sufhydryl group of the polypeptidechain of the protein. Native IGFBP-4 does not possess free cysteinsbecause all cysteins are involved in the formation of disulfide bonds.Reduction of native IGFBP-4 in the presence of mild or lowconcentrations of reducing agents such as β-mercaptoethanol,dithiotreitol or TCEP results in selective opening of a disulfide bondand the exposure of reduced sulfhydryl groups which can be specificallymodified with PEG-maleimide.

IGFBP-4 was dialyzed at a concentration of 0.75 mg/ml against 20 mMsodium phosphate, 150 mM NaCl, pH 7.2 and DTT or β-mercaptoethanol wasadded to concentrations of 30 to 1000 uM. The mixtures were incubatedfor 4 h and than an aqueous solution of 20 kDa PEG-maleimide was addedto a concentration of 1.6 mg/ml. The reaction was stopped and analyzedafter one hour by SDS-PAGE. MonoPEGylated IGFBP-4 was isolated fromsamples containing the highest proportion of this PEGylated derivativeby methods described for random PEGylated IGFBP-4.

It is assumed that the disulfide bonds in the middle domain of IGFBP-4are highly sensitive to reduction and therefore enablescysteine-specific PEGylation at cysteine110 or cysteine117. Thespecificity of the coupling reaction for cysteine 110 and 117 of IGFBP-4was confirmed by peptide mapping of the isolated monoPEGylated IGFBP-4and identification of the peptides by LC-MS mass spectrometry andsequencing of the peptide peaks. PEGylated forms of IGFBP-4 demonstrateda reduced peak area of the peptide containing the two cysteines.

EXAMPLE 5

Purification of 40 kDa-PEG-IGFBP-4 Isomers

Preparative separation of PEGylation products for biochemical andbiological analysis is achieved by size exclusion chromatography on asephacryl S 400 column (Pharmacia) in a running buffer consisting of 20mM sodium phosphate pH 7.5 supplemented with 500 mM sodium chloride.

The 40 kDa-PEG-IGFBP-4 isomers elute earlier in size exclusionchromatography as compared to the unmodified form. This is due to anincreased hydrodynamic radius of the molecule.

Eluting fractions were further analyzed by SDS-PAGE. In SDS-PAGEproteins are separated according to their molecular weight PEGylatedforms of IGFBP-4 migrated more slowly than the wildtype protein. Thespeed of migration is inversely correlated with the amount of PEGattached to the protein. Separation was performed on NOVEX 4-12% NuPagegels in a MOPS SDS buffer system.

Products were combined to three pools which are designated as follows:

-   -   Poly40 kDa-PEG-IGFBP-4: a mixed population of PEGylation        isoforms, consisting of more than 90% IGFBP-4 carrying two or        more 40 kDa mPEG2 residues. The theoretical molecular weight of        the poly40 kDa-PEG-IGFBP-4 is 86 kDa and higher. Apparently they        run at more than 200 kDa in SDS-PAGE    -   Mono40 kDa-PEG-IGFBP-4: a more than 90% homogeneous pool of        PEGylated IGFBP-4 having one molecule 40 kDa mPEG2 attached to        one molecule IGFBP-4. Most probably this pool consists of a        mixture of positional isomers, which means that the PEG chain        may be linked to different amino add residues in individual        protein molecules. The theoretical molecular weight of mono40        kDa-PEG-IGFBP-4 is about 66 kDa. Apparently mono40        kDa-PEG-IGFBP-4 runs at 120 kDa in SDS-PAGE.    -   UnPEGylated IGFBP-4: a homogeneous pool of IGFBP-4 that did not        react with the PEG reagent and is recovered e.g. for recycling.

EXAMPLE 6

a) Production of Random PEGylated IGFBP-4 (20 kDa)

Wildtype human recombinant IGFBP-4 was randomly PEGylated by theaddition of an aqueous solution of N-hydroxysuccinimidyl ester ofmethoxypoly(ethylene glycol)propionic acid, MW 20,000 (ShearwaterPolymers, Inc.; Huntsville, Ala.; thereafter named 20 kDa mPEG-SPA). 20kDa mPEG-SPA was added in a molar ratio of 3 molecules PEG per moleculeIGFBP-4. The reaction was allowed to proceed at room temperature for 30minutes and was finally quenched by adding 1M arginine solution(buffered to pH 8.0 with HCl) to a final concentration of 100 mM.

The outcome of the PEGylation reaction was optimized for maximalproduction of monoPEGylated IGFBP-4 with simultaneous minimalconsumption of mPEG-SPA reagent by carefully titrating protein and PEGconcentrations. For IGFBP-4, yields of the monoPEGylated isoforms isbest at elevated protein concentrations (c=5 mg/ml or higher) and a 1.5molar excess of PEGylation reagent

b) Purification of 20 kDa-PEG-IGFBP-4 Isomers

Preparative separation of PEGylation products for biochemical andbiological analysis was achieved by size exclusion chromatography on aSephacryl S 300 column (Pharmacia) in a running buffer consisting of 20mM sodium phosphate pH 7.5 supplemented with 500 mM sodium chloride. The20 kDa PEGylated species elute earlier in size exclusion chromatography(SEC) as compared to the unmodified form This is due to an increasedhydrodynamic radius of the molecule.

Eluting fractions were analyzed by SDS-PAGE. In SDS-PAGE proteins areseparated according to their molecular weight PEGylated forms of IGFBP-4migrated more slowly than the wildtype protein. The speed of migrationis inversely correlated with the amount of PEG attached to the protein.Separation was performed on NOVEX 4-12% NuPage gels in a MOPS SDS buffersystem.

EXAMPLE 7

Removal of 40 kDa mPEG2 or 20 kDa mPEG-SPA by Ion ExchangeChromatography

Residual PEGylation reagents that did not react with IGFBP-4 wereremoved by ion exchange chromatography (IEC) using SP sepharose(Pharmacia). Samples were dialyzed before loading onto the columnagainst 20 mM sodium phosphate pH 5.5 to reduce the concentration ofsodium chloride and to adjust to the acidic pH. Under these conditions,free PEG did not bind to the column resin and was detected in the columnflow through by a cholorimetric assay as described by Nag, A., et al.,Anal. Biochem. 237 (1996) 224-231. Elution of bound protein wasperformed in a single step with 300 mM sodium chloride in 20 mM sodiumphosphate pH 5.5. Samples were dialyzed against 20 mM sodium phosphatepH 7.5, 150 mM sodium chloride before storage or further analysis.

EXAMPLE 8

Determination of Binding Activities by Size Exclusion Chromatography

Distinct quantities of IGFBP-4 (25 μg or equivalent amounts of PEGylatedisoforms of IGFBP-4) were titrated against known amounts (3,6 and 9 μg,respectively) of IGF-1. Residual free IGF-I was quantified by peakintegration after separating it from IGF-I/IGFBP-4 complexes by sizeexclusion chromatography on an HRP75 column (Pharmacia, running bufferconsisting of 20 mM sodium phosphate pH 7.5 supplemented with 500 mMsodium chloride; flow rate 1 ml/min).

In this assay, unPEGylated, mono20 kDa-PEG-IGFBP-4, poly20kDa-PEG-IGFBP-4 and mono40 kDa-PEG-IGFBP-4 showed identical binding ofIGF-1. E.g. 96 nmol of mono20 kDa-PEG-IGFBP-4 bound 70 nmol of IGF-I.

EXAMPLE 9

Determination of Binding Parameters with FCS

The ability of IGFBP-4 to form complexes with TAMRA(tetramethylrhodamine) labelled IGF-I was measured by FluorescenceCorrelation Spectroscopy (FCS). The assay principle is, that in theabsence of a binding partner free fluorescently labelled IGF-I diffuseswith a distinct velocity. Addition of IGFBP-4 or PEGylated isoformsleads to complex formation with the labelled IGF-I and a concomitantchange in its diffusion velocity. Due to the different diffusionbehavior FCS can differentiate bound from freeIGF-I and quantify itDetermination of the amount of bound IGF-I for several concentrations ofIGFBP-4 allows one to set up a binding curve and to determine kDa valuesby curve fitting.

All measurements were performed on a Confocor I (Zeiss, Jena) at awavelenght of 543 nm in a buffer consisting of 100 mM HEPES (pH 7.6),120 mM NaCl, 5 mM KCl, 1.2 mM Mg2SO4; 1 mM EDTA, 10 mM D(+) Glucose, 15mM sodium acetate, 1% dialyzed bovine serum albumin.

IGF-I binding appeared to be indistinguishable for several IGFBP-4batches; mono20 kDa-PEG-IGFBP-4, poly20 kDa-PEG-IGFBP-4, mono40kDa-PEG-IGFBP-4 and wildtype IGFBP-4 showed comparable behavior in termsof complex formation and binding constants (0.34±0.08 nM).

EXAMPLE 10

Determination of Binding Parameters

Inhibitory constants (IC₅₀ values) for wildtype IGFBP-4 and severalPEGylated isoforms were determined in Biacore experiments(http://www.biacore.com). Briefly, wildtype IGFBP-4 was immobilized to aBiacore CM5 chip surface by NHS-EDC coupling chemistry as known from theart (http://www.biacore.com). All IGF-I binding experiments wereconducted in a commercially available buffer (Biacore HBP-EP; 0.01MHepes pH 7.4; 0.15M NaCl; 3 mM EDTA; 0.005% polysorbat 20 (v/v)). Todetermine IC₅₀ values, 10 nM IGF-I was mixed with eight concentrationsfrom 0.5 to 1000 nM of wildtype IGFBP-4 (or of mono20 kDa-PEG-IGFBP-4 ormono40 kDa-PEG-IGFBP-4) and applied on the chip with immobilizedIGFBP-4. Inhibition was measured as a decrease of response unitscompared to samples of 10 nM pure IGF-I in the absence of IGFBP-4.Mono20 kDa-PEG-IGFBP-4 and mono40 kDa-PEG-IGFBP-4 inhibited IGF-Ibinding as efficient as wild type control IGFBP-4 with IC₅₀ values ofabout 4±2 nM.

EXAMPLE 11

Inhibition of IGF-I Induced IGF-I-Receptor Phosphorylation by PEGylatedIGFBP-4 Isoforms

Confluent monolayers of NIH3T3 cells stably expressing human IGF-IR in3.5 cm dishes were starved in DMEM containing 0.5% dialyzed fetal calfserum. After 48 h, cells were incubated without any hormone or with5×10⁻⁹ M IGF-I; each sample was preincubated with increasingconcentrations of IGF-binding proteins or PEGylated isoforms thereof atroom temperature for 1 h. After a 10 min stimulation at 37° C., themedium was removed and cells were lysed with 250 μl of lysing buffer (20mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Nonidet P40, 1.5 mMMgCl₂, 1 mM EGTA (1,2-bis(2-aminoethoxyetna)-N,N,N′,N′tetraacidic acid,Aldrich, USA), 10 mM sodium orthovanadate, and protease inhibitorcocktail Complete (Roche Diagnostics GmbH, DE) for 10 min on ice.Subsequently, cells were scraped off the plate and the insolublematerial was separated by centrifugation for 20 min at 4° C. The proteinconcentration of the supernatant was determined using Bicinchoninic acid(Pierce, Rockford, USA; Shihabi, Z. K., and Dyer, R. D., Ann. Clin. Lab.Sci. 18 (1988) 235-239). Equal protein concentration was incubated withthe SDS sample buffer (63 mM Tris-HCl, pH 6.8, 3% SDS, 10% glycerol,0.05% bromphenolblue, 100 mM DTT), boiled for 5 min and loaded on a 7.5%SDS polyacryide gel. After electrophoresis the proteins were transferredon a nitrocellulose membrane which first was blocked for 1 h with the 3%BSA containing PBST (phosphate buffered saline-Tween), then overnightincubated with 1 μg/ml monoclonal anti-phosphotyrosine antibody (RocheDiagnostics GmbH, DE) in PBST that contained 3% BSA. Unbound antibodywas removed by extensive washing. The blot was then incubated with1:10000 diluted anti-mouse IgG-specific antibody conjugated with horseraddish peroxidase (Roche Diagnostics GmbH, DE) and developed.

IGFBP-4, mono20 kDa-PEG-IGFBP-4, poly20 kDa-PEG-IGFBP-4 and mono40kDa-PEG-IGFBP-4 each displayed equally good inhibitory potential. Athree fold molar excess (lowest dose measured) of either isoform blockedreceptor phosphorylation induced by 2 nM IGF-I completely.

EXAMPLE 12

Inhibition of the Growth of Tumor Cell Lines by IGFBP-4 Derivatives

The human tumor cell lines PC-3, MDA-MB 231, DU-145, HT29, AsPC-1 andPancTu-1 (from ATCC, American type culture Collection, Rockville, Md.,U.S.A.) were used to investigate the inhibitory effects of IGFBP-4derivatives on tumor cell growth. 4000 AsPC-1 cells or 1000 cells of theother cell types were seeded per well in 100 ul RPMI medium containing10% FBS (fetal bovine serum) and 1% glutamine. The cells were culteredin the absence or in the presence of unmodified IGFBP-4 or mono20kDa-PEG-IGFBP-4 for 5 days and cell proliferation was quantified bydetecting the cleavage of tetrazolium salts added to the growth medium.Tetrazolium salts are cleaved by mitochondrial dehydrogenase in viablecells (WST-1 assay, PanVera, USA). The growth of the cell lines PC-3,MDA-MB 231, DU-145 was not significantly inhibited by IGFBP-4derivatives but the growth of the cell lines HT29, AsPC-1 and PancTu-1was inhibited up to 55% by IGFBP-4. PEGylated IGFBP-4 is even morepotent than unmodified IGFBP-4. TABLE 1 Inhibition of the growth oftumor cell lines by IGFBP-4 derivatives MDA-MB PC-3 231 DU-145 HT29AsPC-1 Panc-Tu1 IGFBP-4 10 0 0 30 40 50 [% Inhibition] Mono20kDa- 10 010 40 50 55 PEG-IGFBP-4 [% inhibition]

EXAMPLE 13

Serum Kinetics of IGFBP-4 Derivatives

SCID mice were injected subcutaneously with a single dose of 1 mg/200 μlmono20 kDa-PEG-IGFBP-4 or mono40 kDa-PEG-IGFBP-4 or unPEGylated IGFBP-4in PBS. Serum samples were collected in a time range from 0.5 to 120 hafter injection and analyzed for mono20 kDa-PEG-IGFBP-4 or mono40kDa-PEG-IGFBP-4 or unPEGylated IGFBP-4 by Western Blotting with ananti-IGFBP-4 antibody (United Biomedical Inc., USA) after affinitypurification or by ELISA. Affinity purification was performed bycoupling biotinylated IGF-I to streptavidin coated magnetic beads (RocheDiagnostics GmbH, DE) and precipitating IGFBP-4 derivatives out of therespective serum samples by magnetic separation. Bound protein waseluted by heating in SDS sample buffer and separated by SDS PAGE.Proteins were transferred to a PVDF-membrane and detected by an IGFBP-4specific antibody. Quantification of bands corresponding to IGFBP-4derivatives was performed by a Lumilmager device (Roche).

ELISA testing was performed by capturing PEGylated proteins with abiotinylated monoclonal antibody against PEG (Cheng T. et al.,Bioconjugate Chem. 10 (1999) 520-528) bound to a streptavidin coatedmicrotiter plate and specifically detecting IGFBP-4 with a polyclonalIGFBP-4 antiserum (labeled with peroxidase) produced from rabbits.

Serum levels of mono40 kDa-PEG-IGFBP-4 peaked after 24 hours (>75 μg/ml)and remained elevated for up to 120 h. In comparison, unPEGylated ormonoPEG₂₀-IGFBP-4 showed substantially lower peak levels (12 or 35μg/ml, respectively) and a much faster clearance. UnPEGylated IGFBP-4levels returned to baseline after already 2 hours. AUC is significantlyincreased by PEGylation and PEGylation with 40 kDa PEG results insignificantly higher serum levels for a longer period of time thanobserved for the 20 kDa PEG derivative of IGFBP-4.

Daily application of mono20 kDa-PEG-IGFBP-4 or mono40 kDa-PEG-IGFBP-4 orunPEGylated IGFBP-4 led to accumulation in serum of treated mice ofmono40 kDa-PEG-IGFBP-4 only. Serum levels of over 300 μg/ml wereachieved at the end of a three-week study.

EXAMPLE 14

Antitumorigenic Effect of IGFBP-4 Derivatives in the PancTu-1 OrthotopicPancreas Cancer Model

In vitro expanded PancTu-1 tumor cells were removed (0.05% Trypsin-EDTA)from culture flasks and transferred into 50 ml culture medium (RPMI1640) at the day of injection, washed once (300×g, 10 min), resuspendedin PBS, additionally washed with PBS and filtrated. Cell concentrationand cell size were determined and concentration of cells adjusted withPBS to a cell titer of 6.6×10⁷/ml.

Tumor cells in a volume of 15 μl (=1.0×10⁶ cells) were injected undervisible control into the duodenal lobe of the pancreas through theserosa towards the pancreas tissue of 8-10 weeks old female SCID mice(C.B-17) with a body weight of at least about 20 g. Thereafter thepancreas was then gently relocated to the abdominal cavity and theperitoneum incision dosed using continues suture (4-0 vicryl). The skinwas adapted and dosed with 34 wound clips.

Starting on day seven after inoculation with tumor cells two groups ofanimals with 8 animals per group were treated with either mono20kDa-PEG-IGFBP-4 or mono40 kDa-PEG-IGFBP-4. The i.p administered dailydose of 1 mg protein in 0.2 ml PBS was normalized to the protein contentof the sample by determination of the absorption of the protein moietyat 280 nm. A third group of 8 animals was treated only with PBS. After21 days of treatment blood samples were taken from each animal and theprimary tumor volume and the tumor weight of each animal was determined.The pancreatic tumor marker CA19.9, a carbohydrate antigenic determinantexpressed on a high molecular weight mucin (MUC1) was detected by EIA(ADI, Alpha Diagnostics, Texas, U.S.A.) and Cyfra 21.1 were determinedon Elecsys1010 (Roche Diagnostics GmbH, Germany).

Both tumor markers were significanly reduced by treatment withmonoPEG₄₀-IGFBP-4 but not by treatment with monoPEG₂₀-IGFBP-4.

Chronic administration of monoPEG₂₀-IGFBP-4 did not inhibit tumorgrowth. Mean tumor volume at termination was 287 mm³ and very similar tothe control group receiving only PBS (226 mm³). In contrast, treatmentwith monoPEG₄₀-IGFBP-4 reduced tumor growth. Mean tumor volume wascalculated at 163 mm³. TABLE 2 Effect of treatment of PancTu-1 tumorbearing mice with IGFBP-4 derivatives on the serum tumor marker CA19.9CA19.9 levels (median, Change Group Treatment Application U/ml) (%) CI 2Control i.p. 127.3 4 mono20kDa- i.p. 108.5 (−16%) 0.57-1.24 PEG-IGFBP-46 mono40kDa- i.p. 40.2 (−66%) 0.17-0.79 PEG-IGFBP-4

TABLE 3 Effect of treatment of PancTu-1 tumor bearing mice with IGFBP-4derivatives on the serum tumor marker Cyfra 21.1 Cyfra 21.1 levels(median, Change Group Treatment Application ng/ml) (%) CI 2 Control i.p.19.3 4 mono20kDa- i.p. 22.6 (+11%) 0.63-1.74 PEG-IGFBP-4 6 mono40kDa-i.p. 10.2 (−65%) 0.17-0.82 PEG-IGFBP-4

EXAMPLE 15

Influence of PEGylated IGFBP-4 on Normal Kidney Cells/Kidney Organs

Primary tumors and kidney organs were resected and fixed in formalin.Tumors were median divided in two parts and both embedded in one blockof paraplast. Both kidney organs were processed (longitudinal andvertical cutting) and embedded. Routine histological staining withhematoxylin and eosin was performed on paraffin.

Chronic treatment with mono20 kDa-PEG-IGFBP-4 applied s.c. or i.p.induced moderate to severe histopathological alteration of kidneytissue. Cells belonging to proximal tubules were vacuolated without signof inflammation and necrosis. These findings were not observed afters.c. or i.p. application of mono40 kDa-PEG-IGFBP-4.

LIST OF REFERENCES

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1-9. (canceled)
 10. A conjugate comprising insulin-like growth factorbinding protein 4 and a constituent or constituents selected from thegroup consisting of: (1) a single poly(ethylene glycol) group having anoverall molecular weight of from about 30 to about 40 kDa, and (2) twopoly(ethylene glycol) groups having an overall molecular weight of fromabout 30 to about 40 kDa.
 11. The conjugate according to claim 1,wherein the single poly(ethylene glycol) group is a branchedpoly(ethylene glycol) group.
 12. The conjugate according to claim 1,wherein the two poly(ethylene glycol) groups are branched poly(ethyleneglycol) groups.
 13. The conjugate according to claim 1, wherein only oneof the two poly(ethylene glycol) groups is a branched poly(ethyleneglycol) group.
 14. A conjugate according to claim 1 wherein theconjugate is linked to one or two poly(ethylene glycol) groups by acysteine selected from the group consisting of: (1) cysteine 110; (2)cysteine 117; or (3) cysteine 110 and cysteine
 117. 15. A conjugatecomprising insulin-like growth factor binding protein 4 and aconstituent or constituents selected from the group consisting of: (1) asingle poly(ethylene glycol) group having an overall molecular weight offrom about 30 to about 40 kDa, and (2) two poly(ethylene glycol) groupshaving an overall molecular weight of from about 30 to about 40 kDa,comprising, reacting the insulin-like growth factor binding protein 4with activated (polyethylene)glycol under conditions such that said(polyethylene)glycol is chemically bound to said insulin-like growthfactor binding protein 4 by primary amino groups or thiol groups ofinsulin-like growth factor binding protein
 4. 16. A pharmaceuticalcomposition comprising the conjugate of claim 1 and a pharmaceuticallyacceptable carrier.
 17. A method of treating cancer comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a conjugate comprising insulin-like growth factor bindingprotein 4 and a constituent or constituents selected from the groupconsisting of: (1) a single poly(ethylene glycol) group having anoverall molecular weight of from about 30 to about 40 kDa, and (2) twopoly(ethylene glycol) groups having an overall molecular weight of fromabout 30 to about 40 kDa.